Autoepitope-mapping of the U1-70K protein with human-Drosophila chimeric proteins.
The 70K protein is the major autoantigen for anti-RNP autoantibodies directed against the U1 small nuclear ribonucleoprotein complex particle. The U1-70K protein has been epitope-mapped by various groups, and a major antigenic region of about 70 amino acids has been found which overlaps with the RNA binding motif. Attempts to map the major antigenic region further with smaller cloned fragments or with peptides have been hampered by total loss of, or strongly reduced, antigenicity. Thus the major antigenic region is composed of conformational epitopes and a detailed analysis of particular epitopes has not been possible. In the present work, we examine the antigenicity of chimeric proteins assembled from the highly conserved Drosophila melanogaster 70K proteins grafted with human 70K segments. With this approach, the effects on antigenicity of exchanging particular segments can be assayed with the overall structure of the major antigenic domain kept relatively constant. Our results, supported by depletion experiments, show that residues 99-128 from the human protein are essential for recognition by both human and canine anti-RNP autoantibodies. These residues have to be presented in a manner that allows correct conformational interaction between the different protein domains.
Henriksson
E W
EW
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
Pettersson
I
I
D000595:Amino Acid Sequence; D000818:Animals; D001323:Autoantibodies / Q000096:biosynthesis; D001324:Autoantigens / Q000032:analysis*; D004285:Dogs; D004331:Drosophila melanogaster / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D008947:Mixed Connective Tissue Disease / Q000097:blood / Q000276:immunology; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D017386:Sequence Homology, Amino Acid
D001323:Autoantibodies; D001324:Autoantigens; D011993:Recombinant Fusion Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human